Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

Abstract

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetP A is 7 to 11 times more active than the two tetR promoters ( tetP R1 and tetP R2) combined. We characterized ten mutations in tetP A, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetP A activity in vivo. Unexpectedly, tetP A mutations reduce by two- to fourfold the combined activity in vivo of tetP R1and tetP R2, in spite of their locations outside the −35 and −10 regions of tetP R1 and tetP R2. For two tetP A mutations, the negative effect on tetP R activity was also demonstrated in tetR − tetR R- lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetP A mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.